ß-lactamase expression induces collateral sensitivity in Escherichia coli.

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, ß-lactam resistance genes -encoding ß-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the ß-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile ß-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that ß-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of ß-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

The individual contributions of blaB, blaGOB and blaCME on MICs of ß-lactams in Elizabethkingia anophelis.

BACKGROUND: The blaB, blaGOB and blaCME genes are thought to confer ß-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. OBJECTIVES: To determine the individual contributions of ß-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. METHODS: Scarless gene deletion of one or more ß-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of ß-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. RESULTS: The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. CONCLUSIONS: The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Role of tcaA, a potential target as a ceftobiprole resistance breaker in MRSA ß-lactam resistance.

OBJECTIVES: Using a random forest algorithm, we previously found that teicoplanin-associated gene A (tcaA) might play a role in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactams, which we have investigated further here. METHODS: Representative MRSA strains of prevalent clones were selected to identify the role of tcaA in the MRSA response to ß-lactams. tcaA genes were deleted by homologous recombination in the selected MRSA strains, and antibiotic susceptibility tests were applied to evaluate the effect of tcaA on the minimum inhibitory concentrations (MICs) of glycopeptides and ß-lactams. Scanning electron microscopy, RNA sequencing, and quantitative reverse transcription-polymerase chain reaction were performed to explore the mechanism of tcaA in MRSA resistance to ß-lactams. RESULTS: The MIC of penicillin plus clavulanate decreased from 3 mg/L to 0.064 mg/L and that of oxacillin decreased from 16 to 0.5 mg/L when tcaA was knocked out in the LAC strain. Compared with wild-type MRSA isolates, when tcaA was deleted, all selected strains were more susceptible to ß-lactams. Susceptibility to ceftobiprole was restored in the ceftobiprole-resistant strain when tcaA was deleted. tcaA knockout caused "log-like" abnormal division of MRSA, and tcaA deficiency mediated low expression of mecA, ponA, and murA2. CONCLUSIONS: Machine learning is a reliable tool for identifying drug resistance-related genes. tcaA may be involved in S. aureus cell division and may affect mecA, ponA, and murA2 expression. Furthermore, tcaA is a potential resistance breaker target for ß-lactams, including ceftobiprole, in MRSA.

Occurrence of beta-lactamases in bacteria.

Our study highlights the escalating issue of beta-lactam resistance in nosocomial pathogens, driven by the broad spectrum of antibiotic-degrading enzymes and plasmid exchange. We catalogued known beta-lactamases across 230 bacterial genera, identified 2349 potential beta-lactamases across over 673 genera, and anticipate discovering many new types, underscoring the need for targeted gene analysis in combating resistance. This study also elucidates the complex relationship between the diversity and frequency of beta-lactamase genes across bacterial genera, highlighting the need for genus-specific approaches in combating antibiotic resistance and emphasizing these genes' significant global distribution and host-specific prevalence. We report many transcriptional regulators, transposases and other factors in the genomes of 20 different bacterial isolates, some of which are consistent with the ability of these species to adapt to different environments. Although we could not determine precisely which factors regulate the presence of beta-lactamases in specific bacteria, we found that the proportion of regulatory genes, the size of the genome, and other factors are not decisive. Further studies are needed to elucidate key aspects of this process.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group.

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Transposons Carrying the aacC2e Aminoglycoside and blaTEM Beta-Lactam Resistance Genes in Acinetobacter.

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species.

Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to ß-lactam resistance in Enterococcus faecium.

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3-8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE: The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

ESBL-Escherichia coli extracellular vesicles mediate bacterial resistance to ß-lactam and mediate horizontal transfer of blaCTX-M-55.

OBJECTIVES: Extracellular vesicles (EVs) have become the focus of research as an emerging method of horizontal gene transfer. In recent years, studies on the association between EVs and the spread of bacterial resistance have emerged, but there is a lack of research on the role of EVs secreted by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in the spread of ß-lactam resistance. Therefore, the aim of this study was to investigate the role of EVs in the transmission of ß-lactam resistance. METHODS: In this study, the role of EVs in the transmission of ß-lactam resistance in E. coli was evaluated by the EVs-mediated bacterial resistance to ß-lactam antibiotics test and the EVs-mediated blaCTX-M-55 transfer experiments using EVs secreted by ESBL-E. coli. RESULTS: The results showed that ESBL-EVs were protective against ß-lactam antibiotic-susceptible bacteria, and this protective effect was dependent on the integrity of the EVs and showed dose- and time-dependent effects. At the same time, ESBL-EVs can also mediate the horizontal transmission of blaCTX-M-55, and EVs-mediated gene transfer is selective, preferring to transfer in more closely related species. CONCLUSIONS: In this study, we demonstrated the important role of EVs in the transmission of ß-lactam resistance in chicken ESBL-E. coli, and evaluated the risk of EVs-mediated horizontal gene transfer, which provided a theoretical basis for elucidating the mechanism of EVs-mediated resistance transmission.

Filling knowledge gaps related to AmpC-dependent ß-lactam resistance in Enterobacter cloacae.

Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, ß-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type ß-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.

The LysR-type transcription factor LsrB regulates beta-lactam resistance in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta-lactam antibiotics by utilizing the inducible beta-lactamase AmpC. Our picture on the condition-dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild-type levels by lsrB complementation. By transcriptome profiling via RNA-Seq and qRT-PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR-type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR-mediated regulation of ampC. Our results substantially expand the existing model of inducible beta-lactam resistance in A. tumefaciens by establishing LsrB as higher-level transcriptional regulator.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

The ß-Lactamase Activity at the Community Level Confers ß-Lactam Resistance to Bloom-Forming Microcystis aeruginosa Cells.

Many freshwater cyanobacteria, including Microcystis aeruginosa, lack several known antibiotic resistance genes; however, both axenic and xenic M. aeruginosa strains exhibited high antibiotic resistance against many antibiotics under our tested concentrations, including colistin, trimethoprim, and kanamycin. Interestingly, axenic PCC7806, although not the xenic NIBR18 and NIBR452 strains, displayed susceptibility to ampicillin and amoxicillin, indicating that the associated bacteria in the phycosphere could confer such antibiotic resistance to xenic strains. Fluorescence and scanning electron microscopic observations revealed their tight association, leading to possible community-level ß-lactamase activity. Combinatory treatment of ampicillin with a ß-lactamase inhibitor, sulbactam, abolished the ampicillin resistance in the xenic stains. The nitrocefin-based assay confirmed the presence of significant community-level ß-lactamase activity. Our tested low ampicillin concentration and high ß-lactamase activity could potentially balance the competitive advantage of these dominant species and provide opportunities for the less competitive species, thereby resulting in higher bacterial diversity under ampicillin treatment conditions. Non-PCR-based metagenome data from xenic NIBR18 cultures revealed the dominance of blaOXA-related antibiotic resistance genes followed by other class A ß-lactamase genes (AST-1 and FAR-1). Alleviation of ampicillin toxicity could be observed only in axenic PCC7806, which had been cocultured with ß-lactamase from other freshwater bacteria. Our study suggested M. aeruginosa develops resistance to old-class ß-lactam antibiotics through altruism, where associated bacteria protect axenic M. aeruginosa cells.

New boronate drugs and evolving NDM-mediated beta-lactam resistance.

Taniborbactam and xeruborbactam are dual serine-/metallo-beta-lactamase inhibitors (BLIs) based on a cyclic boronic acid pharmacophore that undergo clinical development. Recent report demonstrated that New Delhi metallo-beta-lactamase (NDM)-9 (differs from NDM-1 by a single amino acid substitution, E152K, evolved to overcome Zn (II) deprivation) is resistant to inhibition by taniborbactam constituting pre-existing taniborbactam resistance mechanism. Using microbiological and biochemical experiments, we show that xeruborbactam is capable of inhibiting NDM-9 and propose the structural basis for differences between two BLIs.

Metabolic reprogramming and altered cell envelope characteristics in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.

Molecular basis of ß-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5.

Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.

Molecular evidence of ß-lactam resistant Staphylococcus aureus in equids with respiratory tract infections: Frequency and resistance modulation strategy.

The emergence of antimicrobial-resistant strains in Staphylococcus aureus (ß-lactam and methicillin-resistant) is an overwhelming issue worldwide. Using the purposive sampling technique, 217 equids samples were collected from district Layyah which were subjected to culturing followed by genotypic identification of mecA and blaZ genes by PCR. This study revealed that by phenotypic methods, a prevalence of 44.24%, 56.25%, and 47.92% was found for S. aureus, MRSA, and ß-lactam resistant S. aureus in equids. While genotypically, MRSA was found in 29.63% and ß-lactam resistant S. aureus in 28.26% of equids. In-vitro antibiotic susceptibility testing against S. aureus isolates harboring both mecA and blaZ genes showed a high resistance against Gentamicin (75%), followed by Amoxicillin (66.67%) and Trimethoprim+sulfamethoxazole (58.34%). In an attempt to re-sensitize the resistant bacteria to antibiotics, a combination of antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) was used which revealed synergistic effect of Gentamicin and Trimethoprim+sulfamethoxazole with Phenylbutazone; and Amoxicillin with Flunixin meglumine. Analysis of risk factors revealed significant association with the S. aureus-associated respiratory infection in equids. Phylogenetic analysis of mecA and blaZ genes showed a high resemblance of study isolate's sequences with each other and variable resemblance with already reported isolates obtained from different samples of neighboring countries. This study reports the first molecular characterization and phylogenetic analysis of ß-lactam and methicillin resistant S. aureus in equids in Pakistan. Moreover, this study will help in the resistance modulation of resistant antibiotics (Gentamicin, Amoxicillin, Trimethoprim+sulfamethoxazole) and provide a good insight into planning an effective therapeutic regime.

Phenotypic and molecular characterization of beta-lactam resistant Multidrug-resistant Enterobacterales isolated from patients attending six hospitals in Northern Nigeria.

Infections caused by multi-drug resistant Enterobacterales (MDR-E) are difficult to treat and cause significant mortality, especially in developing countries. This study characterized the phenotypic and genotypic profiles of 49 randomly selected beta-lactam resistant MDR-E previously isolated from patients being managed in hospitals in Nigeria using whole genome sequencing. The study isolates exhibited 85.5% resistance to 3rd generation cephalosporins and 65.3% resistance to carbapenems. The blaTEM-1B (29, 59.2%), blaCTX-M-15 (38, 77.6%), and blaNDM-1 (17, 51.5%) were the most common penicillinase, ESBL, and carbapenem resistant genes across isolates, respectively. Seventeen (45%) of blaCTX-M-15 was carried on the insertion sequence ISEc9 while blaNDM-1 (11, 64.7%) were associated with ISEc33. None of the 21 plasmids detected were associated with ß-lactamase genes. Higher resistance rates were found in E. coli ST-88 (n = 2) and the high-risk ST-692 (n = 2). For Klebsiella species, the high-risk clones ST-476 (n = 8) and ST-147 (n = 3) predominated and had higher phenotypic resistance rates and higher number of AMR genes. The mechanisms and pattern of antibiotic resistance differ from patterns previously described with isolates harbouring a wide range of AMRGs. The detection of several chromosomally mediated carbapenemases in our study also represents a significant finding that warrants further investigation to better understand its' implications for clinical practice and public health. The selected MDR-Es were found to be pan-susceptible to tigecycline and had very low resistance to fosfomycin, suggesting a potential for these as empiric treatments. A surveillance approach incorporating both conventional laboratory techniques and modern molecular techniques is essential for the comprehensive characterization of the emergence and dissemination of antimicrobial resistance in Enterobacterales infections within Nigeria.

A review of penicillin binding protein and group A Streptococcus with reduced-ß-lactam susceptibility.

With the widespread use of antibiotics, antimicrobial resistance (AMR) has become a global problem that endangers public health. Despite the global high prevalence of group A Streptococcus (GAS) infections and the global widespread use of ß-lactams, ß-lactams remain the first-line treatment option for GAS infection. ß-hemolytic streptococci maintain a persistent susceptibility to ß-lactams, which is an extremely special phenomenon in the genus Streptococci, while the exact current mechanism is not known. In recent years, several studies have found that the gene encoding penicillin binding protein 2X (pbp2x) is associated with GAS with reduced-ß-lactam susceptibility. The purpose of this review is to summarize the current published data on GAS penicillin binding proteins and ß-lactam susceptibility, to explore the relationship between them, and to be alert to the emergence of GAS with reduced susceptibility to ß-lactams.